Methods of using molecular constructs for detection of biochemical reactions

ABSTRACT

The present invention relates to molecular constructs and methods of their use in detecting biochemical reactions. In particular, the invention relates to a molecular construct having a capture portion and a substrate portion, where the capture portion isolates the construct from a sample medium, and the substrate portion enables the construct to be acted upon and undergo a physical change which can be detected and measured. These molecular constructs may be used in diagnostic assays, genetic screening for potential risks of certain diseases in individuals, and drug discovery and toxicogenomics, using high throughput screening of compounds.

FIELD OF THE INVENTION

The invention generally relates to molecular biology and biochemistry.The invention provides molecular constructs and methods of use indetecting biochemical reactions, either individually or as a plurality.

BACKGROUND OF THE INVENTION

Biochemical reactions within cells define cellular function andactivity. These reactions include a complex interplay between receptorligand interactions and enzymatic reactions, which orchestrate thesignaling and activities of a cell. Physiological conditions influencethe activity levels of enzymes and proteins mediating these reactions.

For example, modifications of amino acid residues on peptide chains playan important role in the regulation of protein function in cells.Transfer of phosphate groups, methyl groups or carbohydrates inducesconformational changes in protein substrates, which in turn results inchanges in protein function. These reactions are usually reversible.Protein modification involves particular classes of enzymaticactivities. For example, methylation is generally catalyzed by a familyof enzymes, known as methyltransferases. Protein methylation regulatesmembrane attachment of cytosolic proteins and contributes to preventionof C-terminal proteolytic degradation of peptides. For example, most Gproteins are methylated. The methylated cysteine residue is located ator near the carboxyl terminus of G proteins may facilitate attachment ofthe peptides to the membrane for signal transduction (Rando, BiochimBiophys Acta 1300(1):5-12 (1996), Hrycyna, Pharmacol. Ther.59(3):281-300 (1993)). Histidine at position 73 on several kinds ofactin is also methylated. It has been shown that methylation is requiredfor maintaining proper conformation of actin molecules (Yao, J. Biol.Chem. 274(52):37443-9 (1999)). Methylation of lysine residue in the S-1region of myosin results in decreased ATP binding in myofibrinecontraction (Bivin, Proc. Nat'l Acad. Sci USA 91(18):8665-9 (1994)). Ithas also been shown that methylation of membrane proteins may contributeto the development of cardiovascular disease in diabetic patients(Schaffer, Mol. Cell Biochem 107(1):1-20 (1991)). In addition,methylation reduces protein-RNA interaction in nuclear proteins (Kim,Amino Acids 15(4):291-306 (1998)), and selectively modulates SH3domain-mediated protein-protein interactions (Bedford, J. Biol. Chem,275(21):16030-6 (2000)).

Similarly, carbohydrates can be transferred to side chains of specificasparagine, serine, or threonine residues in many secreted proteins orproteins displayed on cell-surfaces. The transfer of a carbohydrate toasparagine at position 2181 on human coagulation factor V results inimpaired interaction between factor V and phospholipid membranes(Nicolaes, Biochemistry 38(41):13584-91 (1999)). Large carbohydrates,known as polysaccharides, are formed by linking many sugar monomers.

SUMMARY OF THE INVENTION

The present invention relates to molecular constructs having a captureportion and a substrate portion. The capture portion serves to isolatethe construct from the sample being tested, while the substrate portionprovides a reactive site for determining the presence or activity levelof a target molecule of interest. In a preferred embodiment, themolecular construct comprises a peptide-nucleic acid (PNA) as a captureportion and a peptide or protein as a substrate portion. Alternativepreferred substrate portions comprise non-peptide molecules, such as forexample, fatty acids, steroids, sugars, co-factors and other entitieswhich act on or are substrates for target molecules of interest.

The present invention also relates to methods of using the molecularconstructs. Methods for detecting one or multiple enzyme activities areprovided by measuring the modified substrate portions of the molecularconstruct, which in turn is indicative of one or more enzyme activitiesin a sample. Similarly, methods for detecting analytes in a cell, a cellpopulation or in a tissue sample are also provided.

Molecular constructs of the present invention having a peptide-nucleicacid (PNA) capture portion are particularly beneficial over those in theprior art. Hybridization of PNA to complementary nucleic acid sequencesare more specific and occur with higher affinity because of theuncharged nature of the PNA backbone. PNA-nucleic acid sequencehybridization is much better because of various PNA characteristics thatnucleic acids, such as DNA or RNA, do not possess. PNA is stable over awide range of pH and low ionic strength conditions. These propertiesmake PNAs especially suitable for use in the detection assays of thepresent invention.

There is a need for an assay for detection and analysis of biochemicalreactions that is accurate, both time and cost efficient, and capable ofscreening one or a plurality of reactions with great sensitivity.Therefore, it may be useful to have a method to detect one or morebiochemical reactions that is easy to use, highly specific, accurate,and sensitive for screening biochemical reactions in order to preventand/or treat diseases, preferably human diseases that may be inducedvirally, bacterially, and parasitically.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an example of detecting microparticles using methods byLEAPS or direct deposition, followed by detection on the arrays usingREAD, flow cytometry, and precipitation methods.

FIG. 2 shows a schematic illustration of PNA-analyte chimerae used inmultiplexed assays immobilized to a carrier's surface.

FIG. 3 shows detection of modified analytes/substrates by antibodies.Modified analytes captured on a solid surface may be detected byfluorescent (f) or peroxidase (E) labeled antibodies in the oneantibody—(A); two antibody—(B); or three antibody—(C) formats.

FIG. 4 shows the MAP kinase signal transduction pathway and reporterchimerae used to identify activity of specific kinases in the MAP kinasepathway.

FIG. 5 shows a schematic illustration of a cell based assay usingPNA-STAT chimerae to determine kinase activity in JAK kinase-mediatedsignal transduction pathways.

FIG. 6 shows a schematic illustration for detecting specific kinaseactivity for PKD1 and Akt1/PKB kinases (A), and may be used to analyzethe Akt/PKB signaling pathway in cell-based assays (B).

FIG. 7 shows a schematic illustration for detecting multiple caspaseactivities using PNA-caspase chimerae.

FIG. 8 shows a schematic illustration for a method of identifying newdrugs targeting GPCR using PNA-small molecule chimerae in cell-basedassays.

FIG. 9 shows a schematic illustration of PNA chimerae hybridized tocomplementary oligonucleotides on color-encoded microparticles detectedby flow cytometry.

FIG. 10 shows a method of using PNA-antibody chimerae for detecting andisolating cells having specific proteins displayed on their cellsurface.

FIG. 11 shows two different approaches for immobilizing oligonucleotideprobes to a solid surface, indirect coupling using Biotin-NeutrAvidinbinding (A) and direct coupling using amine-tosyl reaction (B). Resultsfor titration of biotin-NeutrAvidin mediated indirect coupling ofoligonucleotides onto solid surfaces and of direct conjugation ofamine-tosyl direct coupling of oligonucleotides onto solid surfaces areshown under the appropriate diagram.

FIG. 12 shows high-pressure liquid chromatography purification of twotypes of biotinylated PNA oligomers.

FIG. 13 shows a purified tetramethyl rhodamine (TMR) labeled PNA-peptidechimera (A) and a Cy5-labled PNA-small molecule chimera (B) and theirrespective spectrophotometric profiles.

FIG. 14 shows the results of on-chip hybridization assays testingsequence-specific capturing of biotinylated bis-PNA oligomers to variousoligonucleotides immobilized on microparticle arrays.

FIG. 15 shows the results of in-tube hybridization assays (A) comparedto on-chip hybridization assays (B) for testing sequence-specificcapturing of bis-PNA oligomers to DNA oligonucleotides immobilized oncolor-encoded microparticles.

FIG. 16 shows the results of on-chip hybridization assays testingsequence-specific capturing of bis-PNA-protein chimerae to DNAoligonucleotides (18 bases in length) immobilized on microparticlearrays.

FIG. 17 shows the results of PNA oligomers hybridized to complementaryoligonucleotides immobilized on color-encoded magnetic particles, whereChip B (B) is the negative control for Chip A (A).

FIG. 18 shows the results of on-chip hybridization assays for testingsequence-specific capturing of bis-PNA-small molecule chimerae to DNAoligonucleotides (10 bases in length) immobilized on microparticlearrays, where Chip 2 is the negative control for Chip 1.

FIG. 19 shows the results of three biotinylated phosphorylated peptidestandard curves (Peptide-P-1, Peptide-P-2, and Peptide-P-3) atincreasing concentrations, where the peptides are coupled to specificcolor-encoded beads coated with NeutrAvidin using one microparticlearray.

FIG. 20 shows the results of two biotinlyated phosphorylated peptidestandard curves (Peptide-P-1 and Peptide-P-2) at increasingconcentrations, where the peptides are coupled to specific color-encodedbeads coated with NeutrAvidin.

FIG. 21 shows the results of immunodetection of phosphorylated peptides(Ty-P-1 and Peptide-P-2) using tyramide reagents on microparticle arrayThe monoclonal antibody used in the assay is labeled with peroxidasespecific for phosphorylated modification.

FIG. 22 shows the results of immunodetection of phosphorylated peptides(Ty-P-1 and Peptide-P-2) using phosphorylated peptide-specificmonoclonal antibody on microparticle array. The bound monoclonalantibody is detected by using Cy-5 labeled secondary antibody.

FIG. 23 shows the results of simultaneous immunodetection of multiplephosphorylated peptides using fluorescent-labeled antibodies onmicroparticle arrays.

FIG. 24 shows the general design of a dual-labeled PNA-analyte chimeraand ratio quantification of dual-assay signal detection (A); a specificPNA-peptide chimera labeled with a fluorescent dye and an internal His6tag for determining the ratio quantification of Caspase 3 activity (B);and the results of Caspase 3 activity as determined by ratioquantification (C).

FIG. 25 shows the results of detecting PNA chimerae hybridized oncolor-encoded microparticles by flow cytometric analysis. In FIG. 25A,two green beads (Green A and Green B) are coupled with two types ofoligonucleotides (A) and (B). The PNA chimerae specifically binds tooligonucleotides on the Green B beads, which are detectable on theorange channel. Similarly, in FIG. 25B, the results show that PPNAchimera sequence specifically binds to one type of beads detected usingflow cytometry.

FIG. 26 shows the general design of PNA-peptide chimeric substrates fordetermining protease activity, where the functional domains of thechimerae are labeled with one fluorescent dye (I), two fluorescent dyes(II), and one fluorescent dye with an internal His6 tag (III).

FIG. 27 shows the results of determining caspase activity usingbiotinylated Caspase 3 peptide substrate with end-labeled fluorescentdye on microparticle arrays.

FIG. 28 shows the results of a 2×2 PNA chimera competition assayperformed to test specificity of PNA chimerae captured to DNAoligonucleotides (10 bases in length) on microparticle arrays. Chip 1shows the results of a biotinylated PNA clamp (Biotin-PNA-Clamp) (bottompanel) and a bis-PNA-peptide chimera conjugated with tetramethylrhodamine (Bis-PNA-peptide-TMR) (top panel) captured to color-encodedbeads functionalized with oligonucleotides with a defined base sequence.Chip 2 shows the negative control corresponding to the Biotin-PNA-Clamp(bottom panel) and Bis-PNA-peptide-TMR (top panel).

FIG. 29 shows the orange and green images used for decodingcolor-encoded microparticles, orange and green beads, coupled with asynthetic non-phosphorylated peptide and its correspondingphosphorylated peptide, respectively (left panel). The phosphorylatedpeptides on the Green beads were detected by using Cy-5-labeledantibodies on the chip (right panel).

FIG. 30 shows an illustration of a DNA-peptide chimera (A) and thehybridization results of a DNA-peptide chimera to complementary PNAcoupled on color-encoded beads on microparticle arrays (B).

FIG. 31 illustrates the steps in a multiplexed assay using PNA chimericsubstrates.

FIG. 32A illustrates a multiple substrate assay for kinase using theconstructs disclosed herein.

FIG. 32B illustrates a multiple substrate assay for phosphate using theconstructs disclosed herein.

FIG. 32C illustrates a multiple substrate assay for caspase using theconstructs disclosed herein.

FIG. 32D illustrates a multiple substrate assay binding assay using theconstructs disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a molecular construct comprising a captureportion and a substrate portion. The capture portion allows theconstruct to be isolated from a mixture of molecules, and preferablycomprises a nucleic acid or nucleic acid derivative. The substrateportion allows the construct to be acted upon and undergo a change,which can be measured or detected. The change may be a physical one suchas a chemical modification or a cleavage or addition to the substrateportion. Alternatively the change may be the binding of a ligand or cellto the substrate portion of the construct.

The molecular constructs of the invention may be PNA-peptide chimeraecomprising covalently-linked components, including a PNA oligomer, andone or more derivative forms of PNA (such as for example pPNA), alinker, and a synthetic peptide or other substrate portion. The PNAportion of the chimera (which can be linear or in bis-format) serves asan anchor for capturing the complex to a complementary oligonucleotide,which is immobilized on a solid support. The base-sequence of the PNAanchoring domain provides a unique tag to the analyte of the substratedomain of the chimera. The solid support may have defined physicalcharacteristics. In addition, the solid support may be optically orchemically encoded.

The substrate portion of the chimera serves as a functional/substratecomponent participating in biochemical assays. The substrate portion ofthe molecular construct comprises a molecular structure, which acts as aligand or enzyme substrate in a biochemical reaction. For example,proteins or peptides may be used as a substrate for enzymatic reactionsor provide ligand-binding sites. Peptides may also be useful indetecting ligand-receptor reactions. Proteins may be used as substratesto isolate cell-populations having specific cell-surface markers.Alternatively, enzyme cofactors may form all or part of the substrateportion of the chimera, such as, for example, ATP, cAMP, GTP or thelike. Similarly, fats, steroids and/or fatty acids may form thesubstrate portion of the chimera, in that these molecules also may bemodified during biochemical reactions and thus may be measured using thepresent invention. Carbohydrates may also form all or part of thesubstrate portion of the chimera. These molecules include, for example,simple sugars, starches, polysaccharides, proteoglycans, and the like.

The substrate portion may be modified to contain one or more moieties,such as detectable labels or enzyme cleavage recognition moieties, suchas phosphate groups, sugar groups, methyl groups, or the like. In oneembodiment, the substrate portion of the chimera contains one or morefluorescent tags. These tags may be combined with the use ofpolyhistidine, biotin, digoxygenin tags or the like. By using asingle-labeled substrate portion, enzyme digestion eliminates the tagfrom the digested substrates. By using a double-labeled substrateportion, enzyme digestion eliminates the quenching dye from thesubstrates resulting in emission of fluorescence from the second dyeremaining on the cleaved product. By using a substrate portion with onedetectable tag plus an internal polyhistidine tag, enzyme digestioneliminates the fluorescent dye. In addition, detectable signal from theuncut substrates can be normalized to detect signal generated from theinternal polyhistidine tag. These embodiments are illustrated in detailin Examples 10, 14, 16, and 17, which demonstrate that such molecularconstructs can provide quantitative information regarding enzymeactivities of interest. In another embodiment, the substrate portion ofthe chimera contains one or more phosphate groups or other moieties ofinterest, which may act as part of the enzyme cleavage site for aphosphatase or other enzymes of interest.

The constructs of the invention can be used in methods for detectingchanges in the substrate portion. Methods disclosed herein can be usedto determine protein-protein interactions, ligand-receptor binding,protein modification, protein expression, and enzymatic activity incell-free and cell-based formats. The method of the invention can alsobe used to isolate cells bearing specific cell surface markers. Thesemethods may be used for a variety of purposes. The constructs may beused in genetic screening analyses, in diagnoses, treatment and/orprevention of disease, in drug discovery and in toxicogenomics assaysfor diseases or conditions, such as but not limited to those related tocancer, parasite infections, neuronal disorders, hematopoieticdisorders, muscoskeletal disorders, cardiovascular disorders, lymphaticdisorders, respiratory disorders, endocrine disorders, andgastrointestinal disorders.

Generally, the methods of use for the molecular constructs of theinvention react the PNA-substrate chimera with proteins, ligands, orenzymes in a test sample. This reaction may be carried out inhomogeneous solution or the capture portion of the chimera may bepre-immobilized on a solid matrix prior to the reaction. This reactionmodifies the substrate portion of the chimera, if the test reactiontakes place on the surface of the sample. If the biochemical reactionwas carried out in solution, the chimera is then captured to anoligonucleotide-functionalized solid support in a sequence-specificmanner. In one embodiment, color-encoded microparticles are used as thesolid support. Microparticles may be assembled using methods known inthe art, such as LEAPS or direct deposition. LEAPS is a technology knownas Light-controlled Electrokinetic Assembly of Particles near Surfacesand is a process for on-demand fabrication of particle arrays formultianalyte molecular analysis. LEAPS is described in detail in U.S.Pat. No. 6,251,691 and WO 97/40385, which are incorporated herein, intoto, by reference. After the biochemical reactions are performed, thechimerae are captured to complementary DNA oligonucleotide sensors onthe solid surface. The identity of the chimerae is then determinedaccording to the code of the carrier's surface (such as the color codeof the beads) in subsequent detection and data analysis. Followingcapture, the chimerae on the surface may be detected directly accordingto methods known in the art. Non-limiting examples of such methodsinclude precipitation reactions, peroxidase or fluorescent dyes, labeledantibody, ligand-binding detection, or flow cytometry. Modifiedsubstrate portions of the molecular constructs may be detected by one ofseveral possible formats known in the art, such as, for example, RandomEncoded Array Detection (READ) (WO 97/40385) of assembled particles,flow cytometric detection of particle suspension, precipitation onspatially defined solid surfaces, or the like.

DNA oligonucleotides displayed on a solid surface serve as sensors forsequence specific capture of the anchoring domain (i.e. capture portion)of the PNA chimerae in multiplexed assays. The oligonucleotides withdefined base sequences may contain other modifications, such as spacersequences, chemical groups, ligand(s) and/or labels, to facilitatecoupling, hybridization and/or detection. The solid surface may alsocontain defined chemical groups for coupling. Coupling ofoligonucleotides to the solid surface may be a direct or indirectcoupling. Specific methods for coupling are known in the art. Inpreferred embodiments, oligonucleotide coupling may be achieved viabiotin-NeutrAvidin mediated indirect coupling, or amine-tosyl mediateddirect coupling.

In one embodiment of the invention, the capture portion of the molecularconstruct comprises a peptide-nucleic acid (PNA). PNAs are analogs ofDNA with a peptide-like backbone. The PNA backbone consists of repeatingN-(2-aminoethyl) glycine residues linked by amide bonds. Unlike DNA andRNA, PNA does not contain any pentose and phosphate groups. Theuncharged nature of the PNA backbone allows hybridization of PNA tocomplementary DNA or RNA sequence with much higher affinity andspecificity than do other nucleic acids materials. In addition, PNA andbis-PNA can also hybridize to DNA in a sequence specific fashion to forma local DNA/DNA/PNA triplex. Characteristics of PNAs, and processes forsynthesis of the molecules have been previously disclosed in, forexample, WO 92/20702, to Buchardt et al., on Nov. 29, 1992, which isincorporated herein in toto, by reference. Since PNA hybridization isvery specific, PNA probes may be shorter than DNA probes inhybridization assays. PNA is stable over a wide range of pH andconditions of lower ionic strength may be used. In addition, PNA is asynthetic compound that does not exist naturally. Therefore, bothproteases and nucleases cannot recognize the polyamide backbone of PNAmolecules. This property makes PNAs especially suitable for use inhybridization assays using cell lysates or cells or tissues.

PNAs of the PNA-substrate chimera are preferably 10-25 bases in length,more preferably about 15 bases. The first one or two bases at theN-terminus of the molecule are spacers conjugated with other molecules.The rest of the PNA oligomer serves as a probe mediating thesequence-specific capture of the chimera to the oligonucleotideimmobilized on a solid support. The PNA portion of the chimera may besingle-stranded or may be a bis-PNA. Bis-PNA, also referred to as a “PNAclamp” contain two stretches of PNA oligomers joined together through aflexible “hairpin” linker. Upon hybridization, the bis-PNA forms atriplex structure with complementary DNA.

PNAs have a much higher sequence-specific binding affinity andspecificity to their complementary DNAs than DNA-DNA and DNA-RNAhybridization. When used in multiplexed assays, the anchoring domains ofPNA chimerae hybridize to their complementary DNA oligonucleotidesensors that have been immobilized on the defined surface of the carrierof interest (FIG. 2). The DNA oligonucleotide sensors preferably containa defined base sequence that is complementary to one of the PNA anchors(i.e. capture portions) of the PNA chimera library. Immobilization ofthe oligonucleotide to the carrier's surface may be accomplishedaccording to methods known in the art, which may be either directlylinked to the surface by means of a chemical reaction, such as areaction between the amine group and a tosyl group, or indirectlycoupled to the surface via linker molecules, such as binding ofbiotinylated oligonucleotides to avidin, streptavidin, or NeutrAvidin onthe surface. The DNA oligonucleotide sensor may contain spacermolecules, labels, tags or other reactive groups to facilitateimmobilization, hybridization, or detection. The carriers preferablycontain defined spatial and color characteristics for decoding of theimmobilized oligonucleotides. In a preferred embodiment, the carrier maycomprise a microparticle. When there are n₃ types of DNA oligonucleotidesensors and n₄ types of color-encoded beads, a library of (n₃×n₄)oligonucleotide-functionalized carriers may be synthesized. Each type ofoligonucleotide-functionalized microparticle preferably captures onlyone type of PNA chimera in the multiplexed assays.

In one embodiment of the invention, the molecular construct comprises aPNA-substrate chimera. This embodiment comprises a PNA conjugated to apolypeptide via a linker molecule. PNA oligomers can be synthesized bymethods known in the art. Examples of such method include Boc chemistry,and Fmoc chemistry (Nielsen, P. E., Egholm, M, Peptide Nucleic Acids:Protocols and Applications, pp. 21-50, Horizon Scientific Press, 1999),which is incorporated herein in toto by reference.

In another embodiment of the invention, the substrate portion of thechimera is preferably a peptide, such as a substrate for an enzyme. Onepreferred class of enzymes particularly suitable for the multiplexedform of the present invention are enzymes which are part of a family ofrelated enzymes, such as kinases, caspases, phosphatases, transferases,proteases, nucleases, and the like. For the non-multiplexed form of thepresent invention, any biochemical reaction which utilizes a substratecan be measured, wherein the substrate or active site can be conjugatedto capture portion of the chimera. For polypeptide substrates, aminoacid sequences for substrate peptides are generally available in theprior art.

In the present invention, one preferred method of conjugationincorporates a cysteine at the terminus of the peptide, e.g. theN-terminus of the peptide during its synthesis. The sulfhydryl group onthe side chain of the cysteine residue may be used in conjugation of thepeptide to a linker molecule during synthesis of the chimera. Inaddition, a lysine may be added to another terminus of the peptide forbiotinylation used for detection in certain multiplexed biochemicalassays. A heterobifunctional cross-linker may be used in the synthesisof a PNA-peptide chimera.Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-(carboxy-6-aminocaproate),also known as LC-SMCC, is one of the heterobifunctional cross-linkersthat reacts with the sulfhydryl group and amine-group of analytes ofinterest. As is known in the art, the NHS ester of the LC-SMCC firstreacts with the amine group on the N-terminus of PNA oligomer, at pH7-8, resulting in activation of the PNA molecule. The maleimide group ofthe LC-SMCC then reacts with the sulfhydryl group of the peptide, pH6.5-7.5, forming a functionalized PNA-peptide chimera. The synthesizedPNA-peptide chimerae are purified and concentrated using various methodsknown in the art. Accordingly, a library of PNA-substrate chimerae maybe synthesized for multiplexed biochemical assays. In a PNA-substratechimera library, each peptide may be conjugated to a unique PNAoligomer. However, a given set of PNA oligomers may be used in theconstruction of several sub-libraries of PNA-substrate chimerae.

Although it is not necessary, a preferred embodiment of the presentinvention uses a linker in the conjugation of PNA and substrate. Thecross-linker appears to provide a more flexible structure, in that itserves as a steric hinge for the PNA and the substrate portion of thechimera. The additional flexibility provided by the linker mayfacilitate enzyme substrate interactions as well as PNA-oligonucleotidehybridization. In addition, nanoparticles may be used as linkers in thesynthesis of PNA chimerae. PNA and peptide substrates may besequentially or simultaneously conjugated to the surface of theparticles. When magnetic nanoparticles are used as linkers, theresulting PNA-substrate chimerae may be purified or enriched on demandin assays.

Detection of the modified PNA-substrate chimera may be accomplished in avariety of ways. If a single type of PNA-substrate chimera is employed,any means of detection known in the art is appropriate. If a multiplexedassay is used, then a plurality of populations of chimerae is used. Inthis assay, the capture portion of a given chimera population preferablycorresponds to a single type of substrate portion so that after thebiochemical reaction is completed, the specific modified chimera can besorted and identified based upon its corresponding sequence, i.e., knownanchor sequence. The capture of these populations of chimerae isaccomplished by way of hybridization of the chimerae to a plurality ofsurfaces, each containing thereon an oligonucleotide which iscomplementary to a given capture portion of a PNA-substrate chimera. Theplurality of surfaces may comprise multiple different solid matrixes, ormay be a single solid surface wherein sections of the surface containspecific individual types of oligonucleotides. Multiple different solidmatrices may be in the form of particles, such as magnetic particles,microparticles, nanoparticles, organic or inorganic polymers, metal orceramic membranes, and the like. These may be modified in some way todistinguish between populations, such as by color, ionic charge, shapeor reflective index, or their chemical or physical characteristics. Theparticles may also be physically separated to distinguish betweenpopulations. If a single solid matrix is preferred, examples includearrays or microarrays, wherein the different populations of chimerae arehybridized to discrete sections of the array.

In one embodiment of the invention, oligonucleotides are immobilizedonto color-encoded microparticles. These oligonucleotides serve asprobes to hybridize the target complementary capture portions of thePNA-substrate chimerae from solution, for detection. Based onsequence-specific hybridization, the color codes of the microparticlescan be used to decode signal from a specific peptide in multiplexedbiochemical assays. The oligonucleotide probes preferably contain 15 to50 nucleotides. A functional group, such as, an amine group mayoptionally be incorporated at the end of the oligonucleotide forimmobilization. Alternatively, the terminal nucleotide of the probes maybe biotinylated or labeled appropriately. Procedures for synthesis ofthese modified oligonucleotides are known in the art. In certainsituations, it may be desirable that the oligonucleotide probes haveunique base sequences.

The surface of the color-encoded microparticles may carry definedchemical groups for immobilization of biomolecules used in multiplexedbiochemical assays. The chemical groups may include, for example, epoxy,tosyl, amine, carboxyl, chloromethyl, aldehyde groups or other chemicalgroups. Specific chemical processes for generating activated chemicalgroups on the surface of particles is known in the arts; e.g. PolymerColloids: A Comprehensive Introduction, edited by Fitch, R. M., AcademicPress (1997). Oligonucleotides, DNA fragments, RNA molecules, syntheticpeptides, recombinant proteins, purified native proteins or the like maybe immobilized to the particles via interaction with the surfacechemical groups according to methods known in the art; e.g. BioconjugateTechniques, edited Hermanson, G. T., Academic Press (1995). Individualtypes of color-encoded microparticles may be conjugated with definedtypes of biomolecules, so that detectable signals from multiplexedbiochemical assays can be decoded from the color of the conjugatedmicroparticles. Functionalization of particles with biomolecules may becarried out in parallel by using an automatic device to minimizebatch-to-batch variation. In addition, oligonucleotide probes may bespotted onto the surface of solid matrices. For example, spottedoligonucleotide arrays may be used to capture PNA chimerae. Furthermore,oligonucleotide probes may be directly synthesized on the surface of thesolid matrix such as high:density oligonucleotide arrays for capturingthe PNA chimerae.

In another embodiment of the invention, a method for capturing a libraryof PNA-substrate chimerae is disclosed. This embodiment captures theselibraries on microparticle arrays. Here, pre-formed planar arrays ofencoded particles are used to capture corresponding PNA-substratechimerae prior to performing on-chip biochemical assays. In anotherpreferred embodiment, encoded particles captured by way of a displayedoligonucleotide to PNA-substrate chimerae are assembled after completionof biochemical reactions.

In one embodiment, as illustrated in FIG. 3, modified chimerae capturedon the carrier's solid surface may be detected by antibodies, as isknown in the art. Modified chimerae may be detected by using modifiedsubstrate-specific antibodies that have been labeled. Antibodies may bederived from different classes or types of immunoglobulin moleculesgenerated in culture media, in human or animal bodies. Signal fromenzyme labeled antibodies, such as, for example, peroxidase labeledantibodies may be detected by using tyramide enhancement methods. Othersimilar labels and enzymes may be used. Such molecules are familiar tothe skilled artisan. Fluorescent-dye labeled antibodies may be used forthe detection of the modified substrates. In addition to single-antibodyand dual antibody detection systems shown in FIGS. 3A and B,respectively, modified chimerae captured on the solid surface may bedetected by using the three-antibody detection system (FIG. 3C).Briefly, primary antibodies bound to the modified substrate may be boundto secondary antibodies that are specific for the primary antibodies.The complex of the modified substrate, the primary antibody and thesecondary antibody may be detected by using a labeled tertiary antibody.Furthermore, modified substrates may be detected by using specificchelating molecules, for example, gallium nitrilotriacetate (galliumNTA)which has high affinity to phosphate groups. Thus, gallium-NTA may beused in the detection of phosphorylated peptide of the PNA chimerae.

Conditions for hybridizing the PNA-substrate chimerae to DNAoligonucleotides on a solid matrix are known in the art; see, e.g.,Peptide Nucleic Acids: Protocols and Application, edited by Nielsen, P.E. & Egholm, M., pg. 87-162, Horizon Scientific Press (1999). Methodsknown in the art provide a wide range of suitable hybridizationconditions and solutions for capturing the chimera to the sensoroligonucleotides. In a preferred embodiment, the process for capturingthe PNA-substrate chimerae to oligonucleotide-functionalizedmicroparticles conforms to the conditions described hereinbelow.Pre-hybridization may preferably incubate theoligonucleotide-functionalized microparticles in a hybridization buffer(e.g. 10 mM sodium phosphate, 15 mM sodium chloride, 1 mg/ml BSA, and0.1 mg/ml heat-denatured herring sperm DNA, 0.1% SDS, 10% formamide, pH7.2), at about 45° C. with constant rotation for about 30 minutes. TheBSA acts to block unoccupied sites on the surface of the microparticlesduring pre-hybridization. Other large non-specific proteins (such asnon-fat dry milk) may be suitable as a substitute for BSA.

The hybridization step preferably utilizes oligonucleotide probes havingfully complementary sequences to the capture portion of the chimera.PNA-substrate chimerae are added into a suspension ofoligonucleotide-functionalized particles to a final concentration of 10to 100 nM. Hybridization may be carried out at appropriate conditions,preferably at about 45° C. for about 1 hour with constant rotating.After hybridization, the hybridized microparticles are preferably washedwith a washing buffer, more preferably, a high stringency buffer (e.g.10 mM sodium phosphate, 15 mM sodium chloride, 0.1% SDS, pH 7.2) atabout 45° C. for about 30 minutes with constant rotating. Themicroparticles are then washed with a first washing buffer (e.g. 10 mMsodium phosphate, 15 mM sodium chloride, pH 7.2), followed by washingwith a second washing buffer (e.g. 100 mM sodium phosphate and 150 mMsodium chloride, pH 7.2) at about room temperature for about 10 minutes.The hybridization conditions and the washing frequency may be adjustedaccording to the base sequence of the capture portion of the chimerae.General protocols for PNA hybridization have been previously disclosedby Nielsen and Egholm (Peptide Nucleic Acids, Protocols andApplications, Edited by Peter E. Nielsen and Michael Egholm, HorizonScientific Press (1999)).

Multiplexed assays of the present invention are particularly well-suitedfor determining the presence or activity levels of related entities,such as members of an enzyme family, related DNA-binding molecules,related receptor-ligand pairs or the like. One preferred family ofenzymes whose activities may be determined in the multiplexed assays ofthe present invention are those that catalyze the phosphorylation and/ordephosphorylation of proteins. Phosphorylation of serine, threonine, andtyrosine residues is generally considered one of the most importantregulatory mechanisms for intracellular events. Stimulation of receptorson cell membranes activate cascades of kinases, resulting inphosphorylation of various intracellular proteins. These reactions arereversible, enabling cells to respond in a dynamic way to a myriad ofstimulatory signals. By using conventional methods, it is difficult tosimultaneously determine activities for multiple kinases in theinterconnected signal transduction pathways. Using the molecularconstructs of the present invention, unique tags for each of multiplesubstrates can be prepared to permit multiplexed analysis. Uponphosphorylation, phosphorylated PNA chimerae, for example, can besequence-specifically captured to oligonucleotide-functionalizedcolor-encoded microparticles for detection using detectable ligandsspecifically reactive with the phosphorylated substrate, such as, forexample, phospho-substrate binding proteins, phospho-substrate-specificantibodies or phospho-substrate binding chelate. Thus, activities ofmultiple kinases can be simultaneously determined in tissues and cellsand other samples.

One example of measuring the activity of a family of kinase proteins isschematically shown in FIG. 4. Here, the Raf proteins (Raf-1, A-Raf, andB-Raf) are serine-threonine kinases with homology to the PKC family,containing an amino-terminal regulatory domain and a carboxy-terminalcatalytic domain. Members of the Raf family bind to Ras proteinsresulting in their translocation to the plasma membrane, and subsequentactivation. By phosphorylation of inactive MEK1 kinase, Raf kinasesactivate MEK1 kinase that, in turn, activates MAP kinase 2/Erk2. Rafkinases mediate the transduction of signals from Ras to MAP kinase.Regulation of these signaling complexes has been recently reviewed byKolch (J. Biochem (2000), 35:289-305).

Activity of MAP kinase 2/Erk2 may be determined by using PNA-MAP kinasesubstrate peptide chimerae. The phosphorylated PNA-peptide chimerae maybe sequence-specifically captured to oligonucleotide-functionalizedcolor-encoded microparticles followed by detection of the phosphorylatedserine/threonine moiety. By adding kinase inhibitors, such as, forexample, 2′-amino-3′-methoxyflavone (C₁₆H₁₃NO₃), specific kinases may beinhibited in the signal transduction pathway (See FIG. 4). Thus, cascadekinase assays may be used with the molecular constructs of the presentinvention to detect kinase activity in cell samples. Such assays arevaluable in detecting aberrant kinase activities associated with diseaseconditions and stages of disease. Many important growth factors useRas/Raf signal transduction pathways to stimulate cells to multiply andthe signaling complexes have been found to have abnormal activity incertain cancers. These assays are also useful in methods associated withdrug discovery and studies of signal transduction.

A second non-limiting example for determining multiple kinase activitiesrelates to the JAK (Janus-family tyrosine kinase) family of non-receptortyrosine kinases. JAKs are associated with cytokine receptors inside thecell, and are activated by extracellular cytokines. JAK kinases (JAK1,JAK2, JAK3 and Tyk2) have a kinase domain and a pseudokinase domainwhich is not enzymatically active. Cytokine binding induces cytokinereceptor dimerization, activating associated JAKs which phosphorylatethe receptor itself. The phosphorylated receptor then serves as adocking site for the SH2-containing STATs (signal transducers andactivators of transcription). JAK1 is essential for signaling from IL-2,IL-6 (gp130), and interferon (IFN) receptors. JAK3 is required forsignaling through cytokine receptors that share a common g chain. JAK2is required for signaling through a different subset of cytokinereceptors. In addition, JAKs also activate the Ras-MAP kinase pathway,and through Tec, the PI-3 kinase pathway in the cell. STATs, theprincipal effectors of JAKs, are latent cytosolic transcription factors.Once phosphorylated by JAK kinases, STATs dimerize in a head-to-tailfashion by way of their SH domains. STAT1 is a critical mediator of IFNsignaling, STAT4 is specific to IL-12, STAT6 is activated primarily byIL-4 and IL-13, while STAT5A and STAT5B appear to be most important inregulation of growth hormone and prolactin. These STAT-inducedphosphorylation events also potentate specific transcription reactions.Regulation of the gp130/JAK/STAT signaling pathways have been reviewedby Heinrich et al. (Biotech. J. 334:297-314 (1998))

A collection of PNA-substrate chimerae of the present invention can beemployed to simultaneously monitor or detect the activity levels ofmultiple JAK or STAT proteins. By using PNA-STAT chimerae,phosphorylation of multiple STATs may be simultaneously determined incell-based assays. A method for measuring multiple STAT protein activitylevels begins by adding various kinds of PNA-STAT chimerae, includingcell-invading PNA chimerae, into a cell culture of interest. In responseto stimulation from a cytokine or other compound of interest, JAK-STATsignal transduction pathways are activated, resulting in phosphorylationof STAT proteins, including PNA-STAT chimerae. When cells are lysed bystandard means, the PNA-STAT chimerae are released into solution. ThePNA-STAT chimerae may then be sequence-specifically captured tooligonucleotide-functionalized microparticles and detected.Hybridization of PNA chimerae to oligonucleotides on microparticles maybe carried out in a test tube (in-tube hybridization) or on siliconchips with microparticle arrays (on-chip hybridization). This embodimentof the invention is schematically illustrated in FIG. 5.

In addition to PNA-STAT chimerae, other kinds of PNA-protein chimerae,such as, for example, PNA-JAK chimerae, and PNA-peptide chimeraecontaining epitopes of interest may be used in the cell-based functionalassays for determining other effectors of other signal transductionpathways. Such assays are particularly useful for blockingkinase-activated transcriptional activity in order to mediate diseaseconditions. The JAK/STAT signaling pathway in humans has been linked tothe development of certain leukemias. In addition, these assays areuseful in drug discovery assays and functional proteomics assays.

Yet another example of detection of multiple kinase activities relatesto Akt, also referred to as PKB and RAC, which is a protein with anactive role in the regulation and occurrence of apoptosis. The Akt/PKBprotein kinase is activated by insulin and various growth factors. Itfunctions in the Wormannin-sensitive pathway, involving PI-3 kinase.Akt/PKB contains an amino terminal pleckstrin homology (PH) domain thatbinds phosphorylated lipids of the membrane in response to activation ofPI-3 kinases. Akt/PKB is activated by phospholipid binding and PKD1phosphorylation at threonine 308 in the activation loop and at serine473 in the C-terminus region. Akt/PKB functions to promote cell survivalby inhibiting apoptosis by means of its ability to phosphorylate andinactivate several targets, such as Bad, Forkhead transcription factors(FKHR), GSK-3, and Caspase-9. Using a PNA-PKDtide chimera and aPNA-Akt/SGK peptide chimera as substrates, activities of PKD1 andAkt/PKB can be determined simultaneously in tissue and cells. Such amethod comprises adding the PNA peptide chimera to a sample to allowphosphorylation to occur. After phosphorylation, the phosphorylated PNApeptide chimerae may be sequence-specifically captured tooligonucleotide-functionalized color-encoded microparticles, includingcolor-encoded magnetic particles. The captured phosphorylated peptidesmay then be detected using a phosphorylated peptide specific ligand, ora phosphorylated peptide-specific antibody. A schematic illustration ofthis process is shown in FIG. 6A.

In addition, as illustrated in FIG. 6B, Akt/PKB signaling may also besystematically investigated in a PNA chimera cell-based assay. Thismethod comprises adding one or more PNA protein chimerae havingsubstrate portions which are substrates of PKD1/2 and/or Akt/PKB, suchas, for example, PKD, Akt/PKB, Caspase 9, FKHR, Bad, and GSK-3 to a cellsample of interest. At this point, the internal kinase activity isdirectly measured by harvesting PNA chimerae to determine the activationstate and/or levels of these proteins. Alternatively, the cell samplecan be treated with a modulator of the activity, such as insulin orgrowth hormone, for example. Binding of these modulators to the cellsurface receptors may activate PDK1/2 located on the interior side ofthe cell membrane. Activated PDK1/2 phosphorylates Akt/PKB that, inturn, phosphorylates Caspase 9, FKHR, Bad, and GSK-3 proteins. The cellsare then lysed, thereby releasing the PNA-protein chimerae. The PNAchimerae are hybridized to sequence-specificoligonucleotide-functionalized microparticles or other solid matrix. Themodified substrate portion of the chimera is then detected by standardmeans. This assay is schematically illustrated in FIG. 6B. This assay isuseful in detecting aberrant kinase activity associated with diseaseconditions and stages of disease. Previous studies have shown thatmutant presenilin-1 gene induces apoptosis and down regulates Akt/PKB inpatients with Alzheimer's disease (Wehl, et al. J. Neuroscience 19:5360-5369 (1999)). The assay is also useful in diagnosing such diseaseconditions.

Another family of proteins which can be analyzed by the method of thepresent invention are caspases. This family of enzymes is associatedwith the regulation of apoptosis, i.e. programmed cell death.Pro-apoptotic signals such as the release of mitochondrial cytochrome cpromote autocatalytic activation of initiator caspases, such as caspase8, 9 and 10. Once activated, these caspases cleave and activatedownstream effectors such as caspases 3, 6 and 7, which in turn cleavecytoskeletal and nuclear proteins of the apoptotic cells. Otherpro-apoptotic stimuli include binding of Fas ligand and TNF-α to cellsurface receptors, DNA damage and stress to the endoplasmic reticulum(ER) inside the cell. Fas and TNF receptors activate caspases 8 and 10,damaged DNA leads to activation of caspase 9, whereas ER stress leads tothe calcium-mediated activation of caspase 12. This complex scheme isillustrated in FIG. 7. The multiplexed assays of the present inventionusing PNA chimerae provide an effective tool to simultaneously monitorthe effects of signaling events or cell function.

In one embodiment of the present invention, PNA-caspase chimerae areprepared, so that each of the chimerae contains a cleavage site of adefined pro-caspase. The PNA-caspase chimerae also contain at least onelabeled tag, preferably two tags, such as, for example, a fluorescenttag and a polyhistidine tag, for detection. These two tags are locatedon either side of the cleavage site of the peptide substrate. Mixturesof cell-permeable PNA-caspase chimerae are added to the cell sample ofinterest. Pro-apoptotic stimuli, such as TNF-α or Fas ligand, may beadded to selected samples to trigger programmed cell death signals.Activation of the apoptotic pathways results in cleavage of variouscaspase substrates, including the PNA-caspase chimerae. One of thefluorescent dyes contained in double-labeled PNA chimerae is eliminatedby caspase-catalyzed cleavage. When the cells are lysed, the PNAchimerae are released. The released PNA chimerae are then captured ontooligonucleotide-functionalized microparticles. Fluorescence from singletagged-cleaved product or from double-tagged uncleaved product isdetermined and analyzed.

In another embodiment of the invention, the method is useful in theidentification of new drugs. In this embodiment, small molecule drugcandidates are conjugated to PNAs and tested for efficacy. SuchPNA-small molecule chimerae can be used in high-throughput screening ofligands for drug discovery. One example of this method is schematicallyillustrated in FIG. 8. Here, a library of PNA-small molecule chimerae isprepared for screening. Each of the chimerae contains a defined smallmolecule as the substrate domain and a PNA oligomer with known basesequence as the anchoring domain. Mixtures of the PNA-small moleculechimerae are added into cell culture of interest and are permitted tobind chimerae to the target of interest and trigger activation of thetarget cells. FIG. 8 shows G-protein coupled receptors (GPCR), whichtrigger signal transduction. After activation, the effective PNA-smallmolecule chimerae are captured to their complementary oligonucleotidesdisplayed on color-encoded microparticles. Detection may be achieved bydetecting a tag on the small molecule or by binding of a smallmolecule-specific ligand which is detectable. It may also be desirableto strip the captured small molecule-PNAs from the particles byincubation in a solution at a temperature higher than the meltingtemperature of the duplex formed by the capture and anchor domain. Therecovered PNA-small molecule chimerae may then be further analyzed.

In addition to detection by fluorescence microscopy, planar arrays ofPNA chimerae hybridized to complementary oligonucleotides oncolor-encoded microparticles, individual microparticle-displayedPNA-chimerae can be detected by flow cytometry. This embodiment isillustrated in FIG. 9. Accordingly, sequence-specific hybridization ofPNA chimerae to oligonucleotides immobilized on color-encoded particlesmay be carried out in suspension. After hybridization, the particles areincubated with ligands having high binding affinity to modifiedsubstrate portions of the chimerae of interest. In addition, the ligandmay contain a specific fluorochrome for detection. Alternatively,secondary detectable ligands may decorate the primary ligands that bindto the chimerae, ensuring that the emission wavelength of the detectionfluorochrome is distinguishable from the fluorescence of thecolor-encoded particles. For detection of the PNA hybridization,particles are analyzed by flow cytometry. Different types of particlesare identified according to their specific fluorescence. Particleshaving captured PNA chimerae may be identified according to specificfluorescence from the detection fluorochrome.

In yet another embodiment of the present invention, the PNA chimerae areused to detect and isolate specific cells, i.e. cells presentingspecific proteins or other identifying entities on their cell surface.For example, PNA-antibody chimerae are produced, wherein the antibody isspecific for a cell surface receptor of interest. The antibody may belabeled with fluorescent dye in order to monitor the capture process.One example of this method is shown in FIG. 10. In this illustration,CD4+ cells and CD8+ cells are detected and captured. Cy3 and Cy5 arewater-soluble cyanine dyes that are fluorescent labels for proteins,modified oligonucleotides and compounds containing primary amines.

In another embodiment of the invention, the substrate portion of thePNA-chimera comprises a nucleic acid binding domain. In this embodiment,the PNA-chimera is exposed to a detectable nucleic acid, which issuspected to be capable of interacting with the functional domain of thechimera. The bound nucleic acid is then captured onto theoligonucleotide captured on the solid matrix and detected. Thisembodiment may also be multiplexed.

The following examples illustrate production and use of the presentinvention. These examples are offered by way of illustration, and arenot intended to limit the scope of the invention in any manner. Allreferences described herein are expressly incorporated in toto byreference.

EXAMPLES Example 1 Coupling of DNA Oligonucleotides to the Surface ofMicroparticles for Capturing of PNA Chimerae

Two different approaches may be used to attach oligonucleotide probes toa solid surface, e.g., color-encoded microparticles. In the firstapproach, indirect coupling, biotinylated oligonucleotides interact withstreptavidin or its derivatives, conjugated to the solid surface. Theprocess for conjugation of proteins to solid surfaces, such asmicroparticles, is known in the art (Bioconjugate Techniques, edited byGreg T. Hermanson, Academic Press, 1995). As shown in FIG. 11A,biotinylated oligonucleotides were efficiently coupled to NeutrAvidin, amodified form of streptavidin, which has been conjugated to themicroparticles. Briefly, an increasing amount of Cy5-labeledbiotinylated oligonucleotides (0.488, 0.977, 1.953, 3.906, 7.813, 15.63,31.25, 62.5, 125, and 250 nM) were incubated given a fixed number ofcolor-encoded NeutrAvidin-functionalized beads. The coupling reactionwas carried out in PBS at room temperature for 30 minutes. Aftercoupling, unbound oligonucleotides were removed by washing the beadsusing PBST. Unoccupied sites on the surface of the microparticles wereblocked using BSA (10 mg/ml in 100 mM phosphate buffer, pH 7.4). Afterwashing with 100 mM phosphate buffer, pH 7.4, the microparticles may bestored in the storage buffer at 4° C. Different types of beads were thencombined into a test tube for assembly into microparticle arrays. Thearrays were examined under a fluorescence microscope. Results for thetitration series of NeutrAvidin are shown in FIG. 11A. Coupling of 31 nMof biotinylated oligonucleotides may be able to saturate all of thebinding sites on the NeutrAvidin-functionalized beads (3.2 μm diameter)show that, as the oligonucleotide concentration increased, the Cy5signal intensity recorded with 1000 ms integration time increased fromapproximately 100 to 6000. The error bars shown in FIG. 11A representstandard deviations of the means.

Oligonucleotides may also be directly conjugated to the surface of solidcarriers by chemical reaction forming a covalent linkage. An amine groupmay be incorporated to the 5′ terminal end of oligonucleotides accordingto known prior art methods. Conjugation of oligonucleotides totosyl-activated microparticles was achieved in a single step reaction.Examples of amine-tosyl mediated direct coupling are shown in FIG. 11B.Briefly, increasing amounts of Cy5-labeled amine-modifiedoligonucleotides (0.977, 1.953, 3.906, 7.813, 15.63, 31.25, 62.5, 125,250, and 500 nM) were incubated with a given fixed number ofcolor-encoded tosyl-activated beads. Generally, the coupling reactionwas carried out in 100 mM sodium phosphate, pH 7.4 at 37° C. overnight.After coupling, unbound oligonucleotides were removed by washing thebeads using PBST, and the microparticles were incubated with bovineserum albumin (BSA) (10 mg/ml in 500 μl of 100 mM phosphate buffer, pH7.4) to block unoccupied sites on the surface of the particles. Blockingwas performed at 37° C. for 1 hour under constant rotation. Differenttypes of beads were then combined into a test tube for assembly intomicroparticle arrays. The arrays were examined under a fluorescencemicroscope. Results for the titration series in FIG. 11B show that asthe concentration of oligonucleotide increased, the Cy5 signalintensity, recorded at 1000 ms integration increased from about 100 to5000. The error bars, shown in FIG. 11B, represent standard deviationsof the means. Coupling by using 250 nM of amine-labeled oligonucleotidesmay be able to saturate all of the binding sites on the tosyl-activatedcoated beads (3.2 μm diameter). After washing with 100 mM phosphatebuffer, pH 7.4, the oligonucleotide-functionalized microparticles may besaved in storage buffer (100 mM phosphate, 1 mM EDTA, with 0.05% sodiumazide) at 4° C. under constant rotation.

Example 2 Purification of Biotinylated PNA Oligomers

Synthetic PNA chimerae may be purified by liquid chromatography. Anexample of high-pressure liquid chromatography (HPLC) purification oftwo biotinylated PNA oligomers is shown in FIG. 12. The biotinylatedbis-PNA (“PNA clamp”) and linear PNA have molecular weights of 4.3 kDand 7.5 kD, respectively. The PNA oligomers were separated on a gelfiltration column in 10 mM Tris-buffer, pH 7.5, with 125 mM NaCl,monitoring the chromatographic profile at a wavelength of 260 nM.Forty-eight (48) fractions of 0.5 ml eluate were collected at a flowrate of 0.5 ml/min. Two unique peaks, short and tall, were identifiedrespectively, corresponding to a 4.3 KD PNA clamp (Fraction 25) and a7.5 KD linear PNA (Fraction 28). Molecular weights of the purified PNAoligomers were confirmed by mass spectroscopy analysis.

Example 3 Spectrophotometric Profile of Purified PNA-Peptide Chimerae

To verify the labeling of PNA chimera with fluorophores,spectrophotometic profiles of the purified PNA chimerae were recordedusing scanning spectrophotometers. Spectrophotometric profiles for thepurified tetramethyl rhodamine (TMR)-labeled PNA-peptide chimera andCy5-labeled PNA, shown in FIGS. 13A and B, respectively display thedistinct absorption characteristics for the PNA and the fluorescentdyes. The peak in the wavelength range of 250-300 nm corresponds to thePNA with or without peptide, whereas the TMR label (FIG. 13A) isidentified by a peak ranging between 500 and 550 nm, and the Cy5 label(FIG. 13B) by a peak in the 625-675 nm wavelength range.

Example 4 On-Chip Hybridization of Bis-PNA Oligomers to DNAOligonucleotides on Microparticle Arrays

On-chip hybridization assays were performed to ascertain thesequence-specific capture of bis-PNA oligomers to DNA oligonucleotidesimmobilized on microparticle arrays. Briefly, the bis-PNA oligomer was abiotinylated PNA clamp with 12-13 thymine bases on each arm. Prior tohybridization, four types of biotinylated oligonucleotides were coupledto NeutrAvidin-functionalized microparticles, the respectiveoligonucleotides containing an 18-mer oligoadenine (A-18), an unrelatedsequence of 18 nucleotides (N-18), a 10-mer polyadenine (A-10), and anunrelated sequence of nucleotides (N-10). After coupling, all ofoligonucleotide-functionalized microparticles along with a negativecontrol particle containing no oligonucleotide capture probe werecombined into one tube for assembly of microparticle arrays on siliconchips. Chips were first pre-hybridized in a buffer containing 90 mMNaCl, 83 mM guanidine thiocyanate, 8 mM MgCl₂, 17 nM EDTA, 0.1% biotin,0.1% Tween-20, 70 mM Tris-HCl, at pH7.5, at 40° C. for 20 min. Then, thebiotinylated bis-PNA was added into the pre-hybridization buffer to afinal concentration of 200 nM. Hybridization was carried out in ahumidified chamber at 40° C. for 1 hour. The negative control chipreceived no PNA in the hybridization buffer. Upon completion ofhybridization, chips were washed with 100 mM NaCl, 10 mM Tris-HCl, at pH7.5, 0.1% Tween-20, at room temperature for 10 min. For detection of thebiotinylated PNAs hybridized to particle-displayed oligonucleotideprobes, the chips were incubated with Cy5-conjugated streptavidin (20μg/ml) in 100 mM NaCl, 100 mM sodium phosphate, pH 7.5, at roomtemperature for 30 min. After washing with 15 mM NaCl, 10 mM Tris-HCl,pH 7.5, arrays were examined by using a fluorescence microscope. Severalimages were taken from the same chip using different filters that passthrough specific wavelengths. The identity of the particles wasdetermined according to their respective color codes. The assay signalsof the beads from the CY₅ image of the ship were merged with the decodeparticles. Assay signals from the matched A-10 and A-18 and from theunmatched N-10 and A-18 particles were extracted for analysis. Particleswith Cy₅ signals were identified by standard image analysis. As shown inFIG. 14, the bis-PNA clamps specifically hybridize to particlesfunctionalized with 10-mers and 18-mers of polyadenine, whereas thebis-PNA clamps do not hybridize to unrelated nucleotide sequences (N-10or N-18) in contrast, negative control arrays to capture no PNA. Acomputer program was used to automatically align the images from thechip. Fluorescent intensity at different wavelengths was extracted fromthe images and assigned to corresponding particles on the chip. Thefluorescent intensity of the internal dyes of the stained beads was thenclustered.

Example 5 In-Tube Hybridization of Bis-PNA Oligomers to DNAOligonucleotides On Color-Encoded Microparticles

In-tube hybridization assays were performed to ascertainsequence-specific capture of bis-PNA oligomers to DNA oligonucleotideson color-encoded microparticles. Briefly, the bis-PNA oligomer was abiotinylated PNA clamp with 12-13 thymine bases on each arm. Prior tohybridization, four types of biotinylated oligonucleotides were coupledto NeutrAvidin-functionalized microparticles, the respectiveoligonucleotides containing an 18-mer oligoadenine (A-18), an unrelatedsequences of 18 nucleotides (N-18), a 10-mer polyadenine (A-10), and anunrelated unrelated sequence of nucleotides (N-10). Controlmicroparticles contained no oligonucleotides on the surface. Aftercoupling, all of the oligonucleotide-functionalized microparticles werecombined into one tube. The oligonucleotide-functionalizedmicroparticles were first pre-hybridized in a buffer containing 90 mMNaCl, 83 mM guanidine thiocyanate, 8 mM MgCl₂, 17 nM EDTA, 0.1% biotin,0.1% Tween-20, 70 mM Tris-HCl, at pH 7.5, at 40° C. for 20 min. Next,the biotinylated bis-PNA was added into the pre-hybridization buffer toa final concentration of 200 nM. Hybridization was carried out in ahumidified chamber at 40° C. for 1 hour. The negative control wasassayed without PNA in the hybridization buffer. Upon completion ofhybridization, particles were washed with 100 mM NaCl, 10 mM Tris-HCl,pH 7.5, 0.1% Tween-20, at room temperature for 10 min, followed byassembly of microparticle arrays on silicon chips. For detection ofcaptured biotinylated PNAs, chips were incubated with Cy5.5-conjugatedstreptavidin (20 microgram/ml) in 100 mM NaCl, 100 mM sodium phosphate,pH 7.5, at room temperature for 30 min. After washing with 15 mM NaCl,10 mM Tris-HCl, at pH 7.5, arrays were examined by using a fluorescencemicroscope. The identity of the particles was determined according totheir respective color codes. Particles with Cy5.5 signal wereidentified by using computer program. As shown in FIG. 15A, the bis-PNAclamps specifically hybridized to particles functionalized with A-18,but not to C-18 and N-18. Specifically, the bis-PNA clamps specificallycaptured to A-18, produced a Cy5.5 signal of approximately 4750arbitrary units at 1000 ms integration time at the Cy5.5 channel, whileC-18 and N-18 functionalized particles produced a Cy5.5 signal of about500 and 1000, respectively. Cy5.5 signal intensities produced by in-tubePNA hybridization (FIG. 15A) were comparable to those for on-chiphybridization (FIG. 15B) performed as in Example 4.

Example 6 Hybridization of PNA-Protein Chimerae to DNA OligonucleotidesDisplayed on Microparticle Arrays

On-chip hybridization assays were performed to test sequence-specificcapturing of bis-PNA-protein chimerae to DNA oligonucleotidesimmobilized on microparticle arrays. Briefly, the PNA protein chimerawas a conjugate of Cy5.5-labeled streptavidin and biotinylated PNAclamp. The PNA clamp contains 12-13 thymine bases on each array.Comparable amounts of Cy5.5-labeled streptavidin and biotinylated PNAwere used in conjugation which was carried out in PBS at roomtemperature for 30 min. Prior to hybridization, four types ofbiotinylated oligonucleotides were coupled to NeutrAvidin-functionalizedmicroparticles, the respective oligonucleotides containing an 18-meroligoadenine (A-18), an 18-mer of oligocytosine (C-18), and an unrelatedsequence of 18 nucleotides (N-18). After coupling, all of theoligonucleotide-functionalized microparticles along with a negativecontrol particle containing no oligonucleotide capture probe werecombined into one tube for assembly of microparticle arrays on siliconchips. Chips were first pre-hybridized in a buffer containing 90 mMNaCl, 83 mM guanidine thiocynate, 8 mM MgCl₂, 17 nM EDTA, 0.1% biotin,0.1% Tween-20, 70 mM Tris-HCl, pH 7.5, at 40° C. for 20 min. Then, thebis-PNA protein chimerae were added into the pre-hybridization buffer toa final concentration of 200 nM. Hybridization was carried out in ahumidified chamber at 40° C. for 1 hour. The negative control chipreceived no PNA in the hybridization buffer. Upon completion ofhybridization, chips were washed with 100 mM NaCl, 10 mM Tris-HCl, pH7.5, 0.1% Tween-20, at room temperature for 10 min. The chips wereexamined by using a fluorescence microscope. Several images were takenfrom the same chip using different filters that pass through specificwavelengths. The identity of the particles was determined according totheir respective color codes. Particles with Cy5.5 signal wereidentified by standard image analysis as described in Example 4. Asshown in FIG. 16, the bis-PNA protein chimerae specifically hybridize toparticles coupled with 18-mers of polyadenine on the chip (140 Cy5.5signal intensity at 1000 ms integration), as compared to the no PNAcontrol chip. In both instances, the C-18, N-18, and control (none; nooligonucleotides on the surface), only resulted in about 45 units ofCy5.5 signal intensity at 1000 ms integration).

Example 7 On-Chip Hybridization of Biotinylated PNA Oligomers toOligonucleotides Immobilized to Magnetic Color-Encoded Microparticles

On-chip hybridization of target molecules to oligonucleotides probeswere immobilized on color-encoded magnetic particles. Specifically,biotinylated oligonucleotides with known nucleotide sequence (0.4 μM)were coupled to a defined type of color-encoded magnetic particles(approximately 6.7×10⁵ particles) that were coated with NeutrAvidin onthe surface. The coupling reaction was carried out in 0.1 ml couplingbuffer (150 mM NaCl, 0.05 mM ethylenediamine tetra-acetic acid (EDTA),0.5% bovine serum albumin, 0.5 mM Tris-HCl, and 100 mM sodium phosphate,pH 7.2) with vortexing at room temperature for 30 min. After coupling,the particles were collected using a magnet. Unreacted NeutrAvidin siteswere blocked with 0.1% biotin in 150 mM NaCl and 100 mM sodiumphosphate, at pH 7.2 with 0.05% Tween-20 at room temperature for 20 minwith vortexing. After blocking, the particles were washed with 0.2 ml of150 mM NaCl and 100 mM sodium phosphate, at pH 7.2 containing 0.05%Tween-20. This protocol was also applied to couple other biotinylatedoligonucleotides to other types of NeutrAvidin-coated particles.

Several types of color-encoded oligonucleotide-functionalizedmicroparticles, including magnetic particles and non-magnetic controls,were combined into one tube for the assembly of microparticle arraysaccording to methods known in the prior art. In this example, arrayswere formed. 2.5×2.5 mm silicon chips containing an array template of4,000 sites. On-chip hybridization of biotinylated peptide nucleic acid(PNA) oligomers on an array of microparticles-displayed complementaryoligonucleotide capture probes was carried out in 30 microliters ofhybridization buffer (90 mM NaCl, 83 mM guanidine thiocyanate, 8 mMMgCl₂, 17 nM EDTA, 0.02% biotin, 0.1% Tween-20, 70 mM Tris-HCl, pH 7.5)containing biotinylated PNA oligomers at a concentration of 200 nM.Hybridization was performed on a rotating shaker at 40° C. for 60 min.Upon completion of hybridization, the array was washed with 50microliters of 250 mM NaCl, 10 mM Tris-HCl, at pH 7.5, 0.1% Tween-20, atroom temperature for 10 min. Captured biotinylated PNA oligomers werevisualized by incubation of the microparticle array withCy5.5-conjugated Streptavidin (18 micrograms/ml) in 150 mM NaCl and 100mM sodium phosphate, pH 7.2, at room temperature for 30 min. Afterwashing with 15 mM NaCl, 10 mM Tris-HCl, at pH7.5, arrays were examinedunder a fluorescence microscope. Fluorescence emitted from thecolor-encoded microparticles and the Cy5.5-labeled PNA oligomers wasdetermined by using optical filters with specific wavelengths. Themagnetic and non-magnetic particles were decoded according to theircolor codes. Particles with Cy5 signals were identified by standardimage analysis as described in Example 4. Assay results illustrated inFIG. 17 showed that PNAs specifically hybridize to complementaryoligonucleotides displayed on color-encoded magnetic and non-magneticparticles. More specifically, Cy5.5 signal intensity was determined fromfour types of color-encoded microparticles (I, II, III, and IV) from twomicroparticle arrays (Chip A and B, for panels A and B, respectively) inthe on-chip hybridization assay. The type I particles are color-encodedmagnetic particles, whereas types II, III, and IV are three differenttypes of color-encoded particles that are not magnetic. Type I and IIparticles were immobilized with biotinylated oligonucleotides that arecomplementary to the PNA oligomers. Type III particles were coupled witholigonucleotides with unrelated base sequence to the PNA. Type IVparticles had no oligonucleotides on the surface. Chip B served as anegative control for Chip A, which was incubated with hybridization mixwithout the PNA. “n” indicates the number of the particles on the chips.Particles of type I and type II produced respective signal intensitiesof about 2250 and 1500, whereas particles of type III and type IV didnot produce significant fluorescence, about 400 and 250 Cy5.5 signalintensity. The bars represent standard deviation of means.

Example 8 On-Chip Hybridization of PNA-Small Molecule Chimerae toOligonucleotides on Microparticle Arrays

On-chip hybridization assays were performed in order to ascertain thesequence-specific capture of bis-PNA-small molecule chimerae to DNAoligonucleotides displayed on color-encoded microparticle arrays.Briefly, the bis-PNA-small molecule chimera was a conjugate of bis-PNAcontaining ten (10) bases in defined sequence on each arm and a Cy5 dye,previously described in connection with FIG. 13 and Example 3. Prior tohybridization, four types of biotinylated oligonucleotides were coupledto NeutrAvidin-functionalized microparticles, as previously described inExample 1. The respective oligonucleotides containing the followingsequences: a 10-mer of polyadenine (A-10) and 10-mers, eachcomplementary to one of the arms of the bis-PNA oligomers (P-10). Alsoincluded were control microparticles displaying no oligonucleotidecapture probes. After coupling, all of theoligonucleotide-functionalized microparticles along with a negativecontrol particle containing no oligonucleotide capture probe werecombined into one tube for assembly of microparticle arrays on siliconchips. Chips were first pre-hybridized in a buffer containing 90 mMNaCl, 83 mM guanidine thiocyanate, 8 mM MgCl₂, 17 nM EDTA, 0.1% biotin,0.1% Tween-20, 70 mM Tris-HCl, at pH 7.5, at 40° C. for 20 min.Bis-PNA-Cy5 conjugate was added into the pre-hybridization buffer to athe final concentration of approximately 200 nM. Hybridization wascarried out in a humidified chamber at 40° C. for 1 hour. The negativecontrol chip was placed into a hybridization buffer containing no PNAchimerae. Upon completion of hybridization, chips were washed with 15 mMNaCl, 10 mM Tris-HCl, at pH 7.5 followed by examination under afluorescence microscope. The identity of the particles was determinedaccording to their respective color codes. Particles with Cy5 signalwere identified from the beads by standard image analysis as describedin Example 4. Assay results for chips in FIG. 18 show that bis-PNA-smallmolecule chimerae specifically hybridized to particles functionalizedwith complementary P-10 capture probes but not to A-10 particles andcontrol particles, produced hybridization signals (recorded at 500 msintegration time) whereas none of the particle on chips produced asignificant signal. Error bars represent the standard deviations ofmeans.

Example 9 Simultaneous Generation of Standard Curves for ThreePhosphorylated Peptides on Microparticle Arrays

Three different biotinylated phosphorylated peptides (Peptide-P-1, P-2,and P-3. Peptide-P-1: Biotin-CKVEKIGEGT[pY]GVVYK (SEQ ID NO: 1), wherethe tyrosine at amino acid 11 is phosphorylated, is derived from humantyrosine kinase substrate p34cdc2 (Cheng, et al. J. Biol. Chem.267:9248-9256, 1992). Peptide-P-2: Biotin-CEGPWLEEEEEA[pY]GWMDFK-Biotin(SEQ ID NO: 2), phosphorylated on the tyrosine at position 13, is humanGastrin-17 (Baldwin et al, Nature, 301, 435-437, 1983). Peptide-P-3:Biotin-CRRLIEDAE[pY]AARGK, phosphorylated on the tyrosine at amino acid10, is derived from the tyrosine phosphorylation site in p60^(src) fromthe Rous sarcoma virus transforming protein (SEQ ID NO: 3) (Casnelli etal, PNAS 79:282-286, 1982). Peptide-P-1, Peptide-P-2, and Peptide-P-3,in defined concentration 0, 0.0012, 0.0024, 0.0048, 0.0064, 0.008, and0.012 pg/ml, were coupled to color-encoded microparticles that had beenfunctionalized with NeutrAvidin according to as those described inExample 1. All of the peptide-functionalized microparticles were thencombined for assembly into microparticle array according to knownmethods such as those described in Example 1. The assembledmicroparticle array was incubated with monoclonal antibodies specificfor the phosphorylation modification on all of the peptides. For thepurpose of signal amplification, the chip was incubated with fluorescentdye-labeled secondary antibodies specific for the primary monoclonalantibodies bound on the arrays. After washing with PBST, the array wasexamined by using a fluorescent microscope according to known prior artmethods, such as the READ assays. By using this method, threephosphorylated peptide standard curves were generated by using onemicroparticle array (FIG. 19). As the peptide concentration increased,the Cy5 fluorescent signal intensity at 500 ms integration timegenerally increased for all three phosphorylated peptides as well,ranging from about 400 to about 1400 units of Cy5 signal intensity at500 times integration. The error bars shown in FIG. 19 representstandard deviation of the means.

Example 10 Generation of Phosphorylated Peptide Standard Curve onMicroparticle Arrays

Each type of biotinylated phosphorylated peptides (Peptide-P-1 and P-2)as described in Example 9 in defined concentration 0, 0.002, 0.004,0.008, 0.01, and 0.12 μg/ml was coupled to defined types ofcolor-encoded beads that had been coated with NeutrAvidin on the surfaceaccording to known prior art methods, e.g. procedures described inExample 1. Unreacted sites on the surface of the particles were blockedaccording to known prior art methods. The functionalized beads with thesame type of phosphorylated peptides were then combined in the test tubefor assembly into microparticle array according to known prior artmethods, such as LEAPS and direct disposition assembly method previouslydescribed in Provisional Application Ser. No. 60/343,621, filed Dec. 28,2001 and U.S. application Ser. No. 10/192,352 filed Jul. 9, 2002. Theassembled microparticle arrays were incubated with monoclonal antibodiesspecific for the phosphorylation modification on all of the peptides.For the purpose of signal amplification, the primary monoclonalantibodies were further bound by using specific secondary antibodies andfluorescent (Cy5)-labeled third antibodies. After washing with PBST, thearrays were examined by using a fluorescent microscope according toknown prior art methods, such as the READ assays. By using this method,standard curves for the phosphorylated peptides were generated-by usingmicroparticle arrays (FIG. 20). For P-Peptide-1, the Cy5 signalintensity at 500 ms integration increased from about 500 to about 5000units, peaking at a concentration of 0.008 μg/ml. Similarly, forPeptide-P-2, the Cy5 signal intensity at 500 ms integration increasedfrom about 500 to about 4500 units, also peaking at a concentration of0.008 μg/ml. The error bars shown in FIG. 20 represent standarddeviation of the means.

Example 11 Immunodetection of Phosphorylated Peptides by usingPeroxidase-Conjugated Antibodies on Microparticle Arrays

Each type of biotinylated phosphorylated peptides or biotinylatednon-phosphorylated peptides was coupled to defined color-encoded beadsthat had been coated with NeutrAvidin on the surface according to knownprior art methods, e.g. procedures described in Example 1. Density ofthe peptides on the surface of the carriers reaches maximum as describedin Example 1. After coupling, the particles were washed by usingphosphate-buffered saline (PBS; 0.1 M sodium phosphate, 0.15 M sodiumchloride, pH 7.2) with the addition of 0.05% Tween-20 (PBST). Then, allof the peptide-functionalized beads were combined into a test tube forassembly of a microparticle array according to known prior art methods,such as Light-controlled Electrokinetic Assembly of Particles nearSurfaces (LEAPS), and direct disposition assembly method previouslydescribed in Provisional Application Ser. No. 60/343,621, filed Dec. 28,2001 and U.S. application Ser. No. 10/192,352 filed Jul. 9, 2002. Theassembled microparticle array was blocked by using 1% bovine serumalbumin (BSA) in PBST. For immunodetection of phosphorylated peptides onthe arrays, the chip was incubated with peroxidase-conjugated monoclonalantibodies specific for the phosphorylation modification. The incubationwas carried out in a humid chamber for binding of the antibodies to thephosphorylated peptides. The antibody-bound microparticle array waswashed by using PBST followed by detection using peroxidase substratesaccording to known prior art methods, such as using the tyramidereagents from Molecular Probes (e.g. Catalog #T-20912, T-20916, etc).

Results for immunodetection of phosphorylated peptides by using tyramidereagents are shown in FIG. 21. Briefly, each of four types ofcolor-encoded microparticles was coupled with one of two types ofphosphorylated peptides (Ty-P-1 or Peptide-P-2), one type ofnon-phosphorylated peptide (Ty-1), or none, peptide control (PBS) whereTy-1 is Biotin-KVEKIGEGTYGVVYK (SEQ ID NO: 4), Ty-P-1 is Ty-1, but has aphosphorylated tyrosine at amino acid 10 (Biotin-KVEKIGEGT[pY]GVVYK),and Peptide-P-2: Biotin-CEGPWLEEEEEEA[pY]GWMDFK (SEQ ID NO: 2) isphosphorylated on the tyrosine at position 14. Thepeptide-functionalized microparticles were assembled into particle arrayfor immunodetection using alkaline-phosphatase-conjugated monoclonalantibody specific for the modification. The bound antibodies weredetected by using the tyramide reagents. The fluorescent signal from thebeads were detected by using a fluorescent microscope according to knownprior art methods, such as the Random Encoded Array Detection (READ)assay. Briefly, fluorescence emitted from the color-encodedmicroparticles and the analytes were determined by using specificoptical filters. The identity of the particles was decoded according totheir unique color codes. Particles with positive assay signal wereidentified by standard image analysis as described in Example 4. Thefluorescent product, fluorescein isothiocynate (FITC) generated from theakaline-phosphtase-catalyzed chemical reaction was precipitated on thebeads with bound antibodies. FIG. 21 shows higher FITC signal intensityin phosphorylated peptides, 11,000 units at 1000 ms integration forTy-P-1 and 15,000 units at 1000 ms integration for Peptide-P-2, andsignificantly lower signal for non-phosphorylated Ty-1 or control,approximately 700 units at 1000 ms integration. The error bars shown inFIG. 21 represent standard deviation of the means.

Example 12 Detection of Phosphorylated Peptides using FluorescentDye-Labeled Antibodies on Microparticle Arrays

Each type of biotinylated phosphorylated peptides or biotinylatednon-phosphorylated peptides was coupled to defined color-encoded beadsthat had been coated with NeutrAvidin on the surface according to knownprior art methods, such as that described in Example 1. Density of thepeptides on the surface of the carriers reached maximum as described inExample 1. After coupling, the particles were washed by using PBST.Then, all of the peptide-functionalized beads were combined into a testtube for assembly of microparticle array according to known prior artmethods, such as LEAPS, and direct disposition assembly methodpreviously described in Provisional Application Ser. No. 60/343,621,filed Dec. 28, 2001 and U.S. application Ser. No.10/192,352 filed Jul.9, 2002. The assembled microparticle array is blocked by using 1% BSA inPBST. For immunodetection of phosphorylated peptides on the array, thechip is incubated with monoclonal antibodies specific for thephosphorylation modification. The incubation is carried out in a humidchamber for binding of the antibodies to the phosphorylated peptides.The unbound antibodies are washed away by using PBST. For the purpose ofsignal amplification, the chip is incubated with secondary antibodiesspecific for the primary monoclonal antibodies bound on the arrays. Thesignal may be further amplified by incubation of the chips withfluorescent-labeled antibodies specific for the secondary antibodies.After washing with PBST, the array is examined by using a fluorescentmicroscope according to known prior art methods, such as the READassays.

Results for immunodetection of phosphorylated peptides by usingfluorescent-labeled antibodies on microparticle arrays are shown in FIG.22. Briefly, each of four types of color-encoded microparticles iscoupled with one of two types of phosphorylated peptides (Ty-P-1 orPeptide-P-2), one type of non-phosphorylated peptides (Ty-1) asdescribed in Example 11. or none, peptide control (PBS). Thepeptide-functionalized microparticles are assembled into a particlearray. The chip was incubated with phosphorylated peptide-specificmonoclonal mouse IgG antibodies followed by incubation with mouseIgG-specific secondary antibodies, and then Cy5-labeled third antibodiesfor detection using a fluorescent microscope. Fluorescence was detectedby Cy5 signals. FIG. 22 shows higher Cy5 signal intensity inphosphorylated peptides, 15,000 units at 1000 ms integration for Ty-P-1and 15,500 units at 1000 ms integration for Peptide-P-2, andsignificantly lower signal for non-phosphorylated Ty-1 or control,approximately 1400 units at 1000 ms integration. The error bars in FIG.22 represent standard deviation of the respective mean intensities.

Example 13 Detection of Multiple Phosphorylated Peptides usingFluorescent Dye-Labeled Antibodies on Microparticle Arrays

Each type of biotinylated phosphorylated peptides or biotinylatednon-phosphorylated peptides was coupled to defined color-encoded beadsthat had been coated with NeutrAvidin on the surface according to knownprior arts, e.g. procedures described in Example 1. Density of thepeptides on the surface of the carriers reached maximum as described inExample 1. After coupling, the particles were washed by using PBST.Then, all of the peptide-functionalized beads were combined into a testtube for assembly of a microparticle array according to known prior artmethods, such as LEAPS, and direct disposition assembly methodpreviously described in Provisional Application Ser. No. 60/343,621,filed Dec. 28, 2001 and U.S. application Ser. No. 10/192,352 filed Jul.9, 2002. The assembled microparticle array was blocked by using 1% BSAin PBST. For immunodetection of phosphorylated peptides on the array,the chip was incubated with monoclonal antibodies specific for thephosphorylation modification. The incubation was carried out in a humidchamber for binding of the antibodies to the phosphorylated peptides.The unbound antibodies were washed away by using PBST. For the purposeof signal amplification, the chip was incubated with secondaryantibodies specific for the primary monoclonal antibodies bound on thearrays. The signal may be further amplified by incubation of the chipswith fluorescent-labeled antibodies specific for the secondaryantibodies. After washing with PBST, the array was examined by using afluorescent microscope according to known prior art methods, such as theREAD assays.

Results for immunodetection of multiple phosphorylated peptides by usingfluorescent-labeled antibodies on microparticle arrays are shown in FIG.23. Briefly, each of six types of color-encoded microparticles wascoupled with one of four types of phosphorylated peptides (Peptide-P-1,Peptide-P-2, Peptide-P-3, or Ty-P-1), one type of non-phosphorylatedpeptides (Ty-1) as described in Examples 9 and 11., or no peptidecontrol (PBS). The peptide-functionalized microparticles were assembledinto the particle array. The chip was incubated with phosphorylatedpeptide-specific monoclonal mouse IgG antibodies followed by incubationwith mouse IgG-specific secondary antibodies, and then Cy5-labeled thirdantibodies for detection using a fluorescent microscope. Fluorescencewas detected by Cy5 signals. FIG. 23 shows higher Cy5 signal intensityin phosphorylated peptides, approximately 14,000 units at 1000 msintegration for Peptide-P-1, Peptide-P-2, Peptide-P-3, and Ty-P-1, andsignificantly lower signal for non-phosphorylated Ty-1 or control,approximately 1000 units and 500 units at 1000 ms integration,respectively. The error bars shown in FIG. 23 represent standarddeviation of the means.

Example 14 Ratio Quantification of Dual-Labeled Analytes

An analyte may be labeled with two fluorescent dyes. One of thefluorescent dyes labeled the analyte internally or externally accordingto known prior art methods, whereas the other dye was incorporated intothe analyte in biochemical reactions of interest. Fluorescence from theanalyte (analyte signal) and fluorescence from the assay may bedetermined by using a fluorescent microscope according to known priorart methods, such as READ assay. The assay signal related to thereaction efficiency may be evaluated by ratio quantification, that is,the ratio of the assay signal over the analyte signal in the assay. Ageneral design for ratio quantification of dual-labeled analytes isillustrated in FIG. 24A.

A specific design of dual-labeled PNA-peptide chimera is shown in FIG.24B. The PNA-peptide chimera contained a PNA anchoring domain and afunctional domain of synthetic peptide. The synthetic peptide containeda Caspase 3 cleavage site flanked by a fluorescent dye (Cy3) at theC-terminus and a polyhistidine (His6) at the N-terminus. The PNA-Caspase3 dual-labeled peptide chimera is as follows: PNA-Linker-Cys (forconjugation) His His His His His His Asp Glu Val Asp AlaLys-(C₁₈-spacer)-Cy3 (SEQ ID NO: 5).

Caspase 3 digestion (by cutting at the 3′ location on the aspartic acidresidue) of the chimera results in cleavage of the Cy3 fluorescent dye.After digestion, the chimerea were captured to the surface of definedcolor-encoded microparticles via sequence-specific DNA/PNAhybridization. The microparticles with captured PNA-peptide chimeraewere assembled into microparticle array. The His6 tag on the chimeraewas detected by using His6-specific monoclonal antibodies on the chip.The primary monoclonal antibodies were further bound by using specificsecondary antibodies and fluorescent (Cy5)-labeled third antibodies. Cy3fluorescent intensity from the microparticle arrays was related to theportion of the uncut chimerae, whereas intensity of the Cy5 labelscorresponded to the total amount of chimerae captured on themicroparticle arrays. Thus, the relative Caspase 3 activity wasquantified by determining the ratio of the Cy3 and Cy5 ratio in theassay (FIG. 24C). The results determined by ratio quantification showthat as Caspase 3 increases in concentration (0, 0.0625, 0.125, 0.5, 1,2, and 4 ng/μl), the fluorescent Cy3/Cy5 signal ratio decreases fromapproximately 1 to 0.6. The error bars shown in FIG. 24C representstandard deviation of the means.

Example 15 Detection of PNA Chimerae Hybridized to Oligonucleotides onColor-Encoded Microparticles by Flow Cytometric Analysis

An example for detection of PNA chimerae hybridized to oligonucleotideson color-encoded microparticles by flow cytometric analysis is shown inFIG. 25A. Briefly, two types of green color-encoded beads (Green A andGreen B) are coupled with two species of oligonucleotides with definedbase sequence (Green A Biotin-Spacer-AAAAAAAAAA, SEQ ID NO: 6; Green B:Biotin-spacer-AAGGAGAGAA, SEQ ID NO: 7), respectively. Immobilization ofthe oligonucleotides to the beads was carried as described in Example 1,or according to prior known arts. The Green A and B beads have uniqueemission wavelength in the green channel, although emission wavelengthin the orange wavelength is predominantly overlapping for the beads(FIG. 25A). The oligonucleotide-functionalized microparticles were usedin the in-tube PNA hybridization as described in Example 5. The PNAanchoring domain of the biotinylated PNA-peptide chimerae bound to thecomplimentary oligonucleotide sensors immobilized on the Green B beads,but not to the unmatched oligonucleotides on the Green A beads. Afterhybridization, the particles were incubated with R-Phycoerythrinconjugated StreptAvidin (1:100) in 1×PBS. The R-Phycoerythrin conjugatedStreptAvidin bound to biotinylated PNA-peptide chimerae hybridized onthe beads. Detection of the hybridized PNA chimerae was performed byflow cytometric analysis. (Schematic as shown in FIG. 25C). As shown inFIG. 25A, after the assay, the Green B beads gained a significant amountof orange fluorescence corresponding to emission wavelength of theR-Phycoerythrin, indicating hybridization of the PNA peptide chimerae tothe Green B beads in the assay (FIG. 25A). Detection by flow cytometrymay be used on an in-tube pPNA hybridization to complementaryoligonucleotides immobilized on microparticles. One of the two types ofNeutrAvidin-coated microparticles was immobilized with biotin space.A(10), the other type of microparticles was not immobilized with anyoligonucleotide. These two types of beads were incubated withbiotinylated bis-pPNA chimerae. The anchoring portion of the bis-pPNAcontained 12-13 thymine bases on each arm for hybridization.Hybridization and washing in in-tube conditions are as described above.Following hybridization, the beads were incubated with fluorescein(DTAF)-conjugted streptavidin (1:300) in 1×PBS. The fluorescein(DTAF)-conjugated streptavidin was bound to biotinylated pPNA chimerahybridized on the beads. Detection of the hybridized pPNA chimerae wasperformed by flow cytometric analysis.

Example 16 General Design of Substrate Peptides for Determination ofProtease Activity using Microparticle Arrays

PNA-peptide chimeric substrates may be used for determining the presenceof protease activity. Synthetic peptides have been routinely used in thedetermination of protease activity. These PNA-peptide chimericsubstrates were designed in at least one of three formats (FIG. 26).Functional domains of the chimerae were labeled with one fluorescent dye(I), two fluorescent dyes (II), and one fluorescent dye plus an internalHis6 tag (III). Protease digestion by an enzyme capable of cleaving aprotein was detected by using the three differently labeled PNA-peptidechimeric substrates. Examples of such proteases include trypsin,chymotrypsin, thermolysin, papin, pronase, and HIV-1 protease. When thesingle-labeled PNA-peptide chimeric substrates (I) was used, proteasedigestion eliminated the presence of fluorescent dye from the digestedsubstrates. When the dual-labeled PNA-peptide chimeric substrates (II)was used, protease digestion eliminated the quenching dye (Dye 1) fromthe substrates resulting in emission of fluorescence from Dye 2 of thecleaved products. When the PNA-peptide chimera with one fluorescent tagand an internal control tag (III) was used, protease digestioneliminated the fluorescent dye. Fluorescent signal from the uncutsubstrate was normalized to levels of detection signal generated fromthe internal His6 tag as shown in Example 16.

Example 17 Determination of Caspase Activity using Microparticle Arrays

Protease activity, such as Caspase activity, was determined by usingend-labeled, biotinylated Caspase 3 peptide substrate immobilized to asolid surface. Equal amounts of biotinylated Caspase 3 peptide substratewith end-labeled fluorescent dye Cy5 was digested with decreasing amountof purified recombinant Caspase 3 (8, 4.8, 2.88, 1.728, 1.037, 0.62,0.373, 0,224, 0.134, 0.081, 0.048 and 0 microgram/ml) in solution. Afterdigestion, the digested products were coupled to the surface of definedcolor-encoded microparticles that had been coated with NeutrAvidin onthe surface according to known prior art methods. Thepeptide-functionalized microparticles were then assembled intomicroparticle arrays according to the known prior art methods, such asLEAPS, and direct disposition assembly method previously described inProvisional Application Ser. No. 60/343,621, filed Dec. 28, 2001 andU.S. application Ser. No.10/192,352 filed Jul. 9, 2002. Fluorescencefrom the uncut peptide substrates was determined by using a fluorescentmicroscope according to known prior art methods, such as the RandomEncoded Array Detection (READ) assay. Protease digestion eliminatedfluorescent dye from the digested products. As shown in FIG. 27, atitration for Caspase 3 digestion was generated from the assay. The Cy5signal intensity at 1000 ms integration increased from about 2600 to5200 units for a decreasing Caspase 3 concentration ranging from 8 to 0microgram/ml. The error bars shown in FIG. 27 represent standarddeviation of the means.

Example 18 2×2 PNA Chimera Composition Assay on Microparticle Arrays

2×2 PNA chimera competition assays were performed to test specificity ofPNA chimerae captured to DNA oligonucleotides on microparticle arrays inthe presence of other PNA chimerae. Briefly, two types of bis-PNA withdifferent base sequences were used in the assay. One of them wasbiotinylated PNA clamp (Biotin-PNA-Clamp), whereas the other isbis-PNA-peptide chimera conjugated with tetramethyl rhodamine(Bis-PNA-Peptide-TMR). Prior to hybridization, two types of biotinylatedoligonucleotides were coupled to defined type of color-encodedmicroparticles that had been coated with NeutrAvidin on the surfaceaccording to known prior art methods, such as that described inExample 1. Sequences of these oligonucleotides were 10-mers ofpolyadenine (A-10) and 10-mers of defined nucleotide sequence (P-10),respectively. Control microparticles (none) contained nooligonucleotides on the surface. A-10 oligonucleotide was complementaryto PNA of the Biotin-PNA-Clamp, whereas P-10 oligonucleotide wascomplementary to PNA of the Bis-PNA-Peptide-TMR chimera. After coupling,all of the oligonucleotide-functionalized microparticles were combinedinto one tube for assembling microparticle array on silicon chips. Thechips were first pre-hybridized in a buffer containing 90 mM NaCl, 83 mMguanidine thiocyanate, 8 mM MgCl₂, 17 nM EDTA, 0.1% biotin, 0.1%Tween-20, 70 mM Tris-HCl, pH 7.5, at 40° C. for 20 min. Then, similaramounts of the Biotin-PNA-Clamp and the Bis-PNA-Peptide-TMR chimera wereadded into the same hybridization buffer for the chip. Negative controlchip received no PNA in the hybridization buffer. Hybridization wascarried out in a humid chamber at 40° C. for 1 hour. Upon completion ofhybridization, the chips were washed with 100 mM NaCl, 10 mM Tris-HCl,pH 7.5, 0.196 Tween-20, at room temperature for 10 min. For detection ofthe biotinylated PNAs hybridized on the particles, the chips wereincubated with Cy5-conjugated streptavidin (20 pg/ml) in 100 mM NaCl,100 mM sodium phosphate, pH 7.5, at room temperature for 30 min. Afterwashing with 15 mM NaCl, 10 mM Tris-HCl, pH 7.5, the chips were examinedby using a fluorescence microscope. The identity of the particles wasdecoded according to their color codes. Particles with Cy5 signal or Cy3signal for rhodamine were identified by using a computer program asdescribed in Example 4. As shown in FIG. 28, the Biotin-PNA-Clamp wasspecifically captured to particles coupled with 10-mers polyadenine asdetected by Cy5 fluorescence (about 2250 units Cy5 signal intensity at500 ms integration), whereas the Bis-PNA-Peptide-TMR chimera was boundto particles with P-10 oligonucleotides as detected by Cy3 fluorescence(about 4500 Cy3 signal intensity at 500 ms integration) on the chip.

Example 19 Assembly of Peptide-Functionalized Microparticle Arrays byLEAPS

Two types of color-encoded microparticles, Orange and Green beads, werecoupled with a synthetic peptide and its corresponding phosphorylatedpeptide, respectively. Amino acid sequences of the peptides areidentical, except there is a phosphate group on a tyrosine residue ofthe phosphorylated peptide. These two types of peptide-functionalizedmicroparticles were mixed in one test tube. The microparticles wereassembled into microparticle arrays on silicon chips by LEAPS asdisclosed in U.S. Pat. No. 6,251,691. The microparticle arrays were thenincubated with mouse monoclonal antibodies specific for thephosphotyrosine of the peptide. Binding of the monoclonal antibodies wasdetected by using Cy5-labeled goat anti-mouse IgG on the chips asdescribed in Example 12. The chips were then examined using afluorescent microscope. The orange and green images were used fordecoding the microparticles, whereas the Cy5 staining labels the greenbeads immobilized with the phosphorylated peptides. More specifically,the image for the orange colored beads represent non-phosphorylatedpeptides, while the image for the green colored beads representphosphorylated peptides. The resulting assay image shows Cy5 fluorescentstaining of phosphotyrosine-specific antibodies as shown in FIG. 29.

Example 20 Hybridization of DNA-Peptide Chimerae to PNA Coupled toColor-Encoded Beads on Microparticle Arrays

Similar to the PNA-peptide chimera previously described, DNAoligonucleotides may be conjugated to synthetic peptides by usingbifunctional cross-linkers. The resulting conjugate is known as aDNA-peptide chimera. The DNA-peptide chimera may be used inhybridization of complimentary PNA oligomer immobilized on color-encodedbeads on microparticle array. The peptide moiety may contain at leastone tag, such as biotin or fluorescent dyes, for detection of thehybridized DNA-peptide chimera on the beads. In addition, ligands withhigh affinity to the peptide or modification of the peptide may also beused for the detection. The principal of hybridizing a DNA-peptidechimera to a PNA-functionalized microparticle is illustrated in FIG.30A. In the present example, a DNA-peptide chimera is a conjugate of adeoxy-adenine oligomer (dA oligomer) and a biotinylated syntheticpeptide with a bifunctional cross-linker, SMCC, succinimidyl4-(N-maleimidomethyl, cyclohexane-1-carboxylate). The DNA-peptideconjugate was purified using liquid chromatography. The purifiedDNA-peptide chimera was used to hybridize PNA thymine oligomersimmobilized on color-encoded beads on microparticle arrays. Thehybridized DNA-peptide chimera was detected by using Cy3-conjugatedStreptAvidin. The microparticle array was examined by using afluorescence microscope. Fluorescence intensity for the bead color andthe Cy3 hybridization signal was determined from the array. The identityof the microparticles was decoded according to their color codes. Beadswith Cy3 fluorescent dye were detected and identified. As shown in FIG.30B, the DNA-peptide chimerae specifically bind to PNAs with perfectmatch base sequence as indicated by a Cy3 signal intensity at 1000 msintegration of about 1000 units and a unmatched perfect base sequenceresulted in about a 300 units Cy3 signal intensity at 1000 msintegration.

Example 21 Caspase Digestion of Multiple Substrates

Reactivity of caspase against multiple peptide substrates could besimultaneously determined by using PNA-peptide chimerae. In thisexample, three types of PNA-peptide chimerae, I, II and III, weresynthesized. Each of the chimerae contains a unique PNA anchoring moietywith defined base sequence, which is conjugated with specific caspasesubstrate peptide as functional moiety. The functional moieties of PNAchimera I, II and III contains amino acids Asp-Glu-Val-Asp (SEQ ID NO:8), Val-Glu-Ile-Asp (SEQ ID NO: 9), and Ile-Glu-Thr-Asp (SEQ ID NO: 10),respectively. These synthetic peptides are Caspase 3, 6, and 8substrates known from the prior arts. The C-terminus of the peptides waslabeled with defined fluorescent dye. Caspase digestion of the chimericsubstrates results in elimination of the fluorescent dye from the PNAanchoring moieties.

The multi-substrate caspase digestion was set up as follows: Certainamounts of chimera I, II and III were mixed together in one test tube.Aliquots of the chimera mix were added into reaction solutionscontaining Caspase 3, 6, and 8, respectively. Caspase digestion of thechimeric substrates was carried out by incubation of the reactionmixtures in a water bath for certain period of time. Negative control isreaction mix without any caspases. After digestion, hybridizationmixtures were by adding equal amount of hybridization buffer, containingguanidine HCl and detergents, into the reaction mixtures. Thehybridization mixtures were then incubated with pre-assembledmicroparticle arrays.

The microparticle arrays contain four types of color-encoded beadsassembled on silicon chips according to methods commonly known in theart. Three types of the beads were functionalized with oligonucleotideswith defined based sequences, which could bind to PNA-peptide chimera I,II and III, respectively, in sequence-specific manners. In addition tothe encoding colors, the fourth-type of the beads also contains colormatching to the fluorescent dye labeled on the chimeras, which was usedfor normalization of color intensity from different arrays.

After hybridization, nonspecific bindings were washed away from thearrays followed by examination using a fluorescent microscope. The assaysignal, color intensity from the hybridized PNA chimeras, was determinedfrom the beads on the arrays. Identity of the beads was decodedaccording to their predefined color codes. Intensity ratios, ratio ofthe assay signal and signal from the control beads, were determined. Thenegative control reaction should give maximum levels of intensity ratiosin the assay. Caspase activity for particular substrates was expressedas percentage of the residual assay intensity ratios relative to thenegative controls on each chip. The relative percentages for the assayintensity ratios from this study are summarized in following table:

PNA-Peptide Chimera Enzymes I II III Caspase 3 53.48% 82.35% 112.03%Caspase 6 91.80% 41.18% 79.08% Caspase 8 73.77% 69.41% 59.89% The boldnumbers represent more intense digestion than expected in the assay.

Example 22 Flow Chart for Enzyme Assays Using PNA

Multiplexed assays using PNA chimeric substrates disclosed herein haveseveral advantages over the prior known arts. In the PNA chimericconstruct, the PNA anchoring portion plays two important roles inmultiplexed assays. First of all, the base sequence of PNA serves as acode for the conjugated substrates. Secondarily, the PNA oligomers serveas specific anchors for the chimeras that may be captured tocomplementary oligonucleotides displayed on encoded solid surface. Thereis no known protease and nuclease for PNA degradation. Thus, the PNAoligomers will be very stable in tissue lysate. Compared to DNA-DNAhybridization, PNA hybridizes to complementary DNA oligonucleotidesunder very mild conditions. The PNA-DNA complexes are stable in low saltsolution, i.e., conditions not favored for DNA-DNA hybridizations. Thus,multiple PNA chimeric substrates may be added into a common reactionmixture for single or multiple types of enzymatic reactions. Asillustrated in FIG. 31, multiplexed assays disclosed herein may becarried out in a simple format, which includes steps of synthesis of PNAchimeric substrates, performing of multiplexed assays, hybridization tooligonucleotides displayed on color-encoded microparticles, detection ofmodified substrates, image acquisition and data analysis.

Example 23 Multiplexed Assays

As one of the preferred embodiments, PNA-peptide chimeras are the idealsubstrates for multiple-substrate enzymatic reactions, such as kinaseassays (FIG. 32A), phosphatase assays (FIG. 32B), caspase assays (FIG.32C), and ligand binding assays (FIG. 32D). Briefly, a library ofPNA-peptide chimeras may be incubated with a kinase of interest. Afteran in-solution phosphorylation reaction, phosphorylated peptides may bedetermined on the captured PNA chimeras (FIG. 32A). Thus, substratepeptides for the kinase may be identified from the library. Design ofthis assay may be applied to drug discovery for target identificationfor altered kinase activity in disease stages. By usingPNA-phosphorylated peptide chimeras as substrates, a similar design maybe applied to multiple-substrate phosphatase assays (FIG. 32B), whichmay be used in drug discovery to identify specific phosphataseinhibitors for various disease stages. In addition, as shown in Example17, PNA-peptide chimeras can be used to determine caspase activity in invitro assays. As illustrated in FIG. 32C, a library of PNA-peptidechimeras may be incubated with a caspase of interest. Substrate peptidesmay be identified from the library after the digestion. Design of thisassay may be used in identification of caspase inhibitors in apoptoticcells. Furthermore, PNA-peptide chimeras may also be used in bindingassays in the analysis of protein-protein interactions orprotein-nucleic acid interactions. As shown in Panel C (FIG. 32D), alibrary of PNA-peptide chimeras may be incubated with targets ofinterest in ligand binding assays. PNA-peptide chimeras bound withspecific targets may be identified from the library (FIG. 32D).

The above description of various preferred embodiments has beenpresented for purposes of illustration and description. It is notintended to be exhaustive or limiting to the precise forms disclosed.Obvious modifications or variations are possible in light of the aboveteachings. The embodiments discussed were chosen and described toprovide illustrations and its practical application to thereby enableone of ordinary skill in the art to utilize the various embodiments andwith various modifications as are suited to the particular usecontemplated. All such modifications and variations are within thesystem as determined by the appended claims when interpreted inaccordance with the breadth to which they are fairly, legally andequitably entitled.

1. A method of detecting a plurality of enzyme activities in a sample,comprising: (a) adding a plurality of types of PNA-substrate chimerae tothe sample, said PNA-substrate chimerae having a PNA capture portion anda substrate for an enzyme, wherein different types have differentsubstrates capable of reacting with different enzymes but each type hasa particular substrate and a selected PNA sequence capture portion; (b)allowing the enzymes to react with the substrate portions of the chimerapopulation; (c) capturing the reacted PNA-substrate chimerae withdifferent types of oligonucleotides, wherein said different types ofoligonucleotides are displayed on differently encoded microparticles,and wherein said capturing is by annealing of a type of oligonucleotideto a selected PNA sequence capture portion; and (d) detecting thechanges in the substrates of captured reacted PNA-substrate chimerae;(e) decoding the encoded microparticles to identify which substratesreacted and thereby identify the presence of particular enzymes in thesample.
 2. The method of claim 1, wherein the microparticles arecolor-encoded.
 3. The method of claim 1, wherein the PNA-substratechimera contains two tags.
 4. The method of claim 3, wherein the tagsare a fluorescent moiety.
 5. The method of claim 1, wherein thesubstrate portion of the PNA-substrate chimera is a substrate of acaspase.
 6. The method of claim 1, wherein the substrate portion of thePNA-substrate chimera is a substrate of a phosphatase.
 7. The method ofclaim 1, wherein the capture portion of the PNA-substrate chimera is abis-PNA.